Donor T cell STAT3 deficiency enables tissue PD-L1–dependent prevention of graft-versus-host disease while preserving graft-versus-leukemia activity

STAT3 deficiency (STAT3–/–) in donor T cells prevents graft-versus-host disease (GVHD), but the impact on graft-versus-leukemia (GVL) activity and mechanisms of GVHD prevention remains unclear. Here, using murine models of GVHD, we show that STAT3–/– donor T cells induced only mild reversible acute GVHD while preserving GVL effects against nonsusceptible acute lymphoblastic leukemia (ALL) cells in a donor T cell dose–dependent manner. GVHD prevention depended on programmed death ligand 1/programmed cell death protein 1 (PD-L1/PD-1) signaling. In GVHD target tissues, STAT3 deficiency amplified PD-L1/PD-1 inhibition of glutathione (GSH)/Myc pathways that regulate metabolic reprogramming in activated T cells, with decreased glycolytic and mitochondrial ATP production and increased mitochondrial ROS production and dysfunction, leading to tissue-specific deletion of host-reactive T cells and prevention of GVHD. Mitochondrial STAT3 deficiency alone did not reduce GSH expression or prevent GVHD. In lymphoid tissues, the lack of host-tissue PD-L1 interaction with PD-1 reduced the inhibition of the GSH/Myc pathway despite reduced GSH production caused by STAT3 deficiency and allowed donor T cell functions that mediate GVL activity. Therefore, STAT3 deficiency in donor T cells augments PD-1 signaling–mediated inhibition of GSH/Myc pathways and augments dysfunction of T cells in GVHD target tissues while sparing T cells in lymphoid tissues, leading to prevention of GVHD while preserving GVL effects.


Mice
BALB/c (H-2 d ) and C57BL/6 (H-2 b ) mice were purchased from the National Cancer Institute animal production program (Frederick, Maryland, USA). Stat3 fl/fl CD4-Cre C57BL/6 (H-2 b ) breeder were kindly provided by Dr Hua Yu's lab at City of Hope National Medical Centre (Duarte, CA). PD-L1 -/-BALB/c breeders were provided by L. Chen (Yale University, New Haven, Connecticut). PD-1 -/-C57BL/6 (H-2 b ) breeder mice obtained from Dr. Tasuku Honjo's laboratory (Kyoto University, Kyoto, Japan). PD-1 -/-C57BL/6 (H-2 b ) mice were crossed with Stat3 fl/fl C57BL/6 (H-2 b ) mice to generate Stat3 fl/fl PD-1 -/-CD4-Cre C57BL/6 (H-2 b ) mice. Splenocytes from STAT3-S727A C57BL/6 (H-2b) and control mice were provided by Dr. M. Isbell (Department of Pathology, NYU Grossman School of Medicine, NY). Animal breeding and experiments were performed in separate specific pathogen-free rooms, and control and experimental mice were kept in separate cages in the same room at City of Hope Animal Research Center (ARC). In all, 12 light/12 dark cycle, temperatures of 68-75 °F with 30-70% humidity are used. All procedures were performed in the animal facility in compliance with a protocol approved by the City of Hope Institutional Animal Care and Use Committee (IACUC) under IACUC protocol 03008. All Mice were euthanized by CO2 inhalation from compressed gas cylinders in compliance with all ethical regulations.

Induction and assessment of GVHD
WT BALB/c recipients were exposed to 850 cGy total body irradiation (TBI) with the use of a [137Cs] source 8-10 hours before HCT, and then injected intravenously (i.v.) with splenocytes (5.0 x 10 6 ), Thy1.2 + cells (1 x 10 6 or 2.5 x 10 6 ), and T cell-depleted BM (TCD-BM) cells (2.5 x 10 6 ) from C57BL/6 donors. Bone marrow T cell depletion was accomplished by using biotin-conjugated anti-CD4 and anti-CD8 mAbs, and anti-biotin Microbeads (Miltenyi Biotec, Germany), followed by passage through a MACS Column-based cell separation device (Miltenyi Biotec, Germany). Enrichment of Thy1.2 + cells from spleen was accomplished by using mouse anti-CD90.2 microbeads (Miltenyi Biotec, Germany). The purity of enrichment was >98%, whereas the purity after depletion was >99%. The assessment and scoring of clinical signs of acute GVHD and clinical cutaneous GVHD has been described previously (1,2) Histopathology Tissue specimens were fixed in formalin before embedding in paraffin blocks, sectioned, and stained with Hematoxylin and eosin (H&E). Slides were examined at 100x (liver) or 200x (small intestine and colon) magnification and visualized with Zeiss Observer II. Organ GVHD severity was blindly assessed according to a defined scoring system, as described previously (3). Liver GVHD was scored by the severity of lymphocytic infiltrate, proportion of involved tracts and severity of liver cell necrosis; the maximum score is 9. Gut GVHD was scored by mononuclear cell infiltration and morphological aberrations (e.g. hyperplasia and crypt loss), with a maximum score of 8.

Bioluminescent imaging
Mice were inoculated with luciferase-expressing BCL1 cells (BCL1/ Luc + ) by i.p. injection or luciferase-expressing BEL-1 ALL cells (ALL/Luc + ) (4) by i.v. injection on day 0 after HCT. For in vivo imaging of tumor growth, 200μl of 15mg/ml firefly luciferin was i.p. injected (Caliper Life Sciences, Hopkinton, MA), and mice were anesthetized for analysis of tumor cell burden by using an IVIS100 (Xenogen) and AmiX (Spectral) imaging system. Data were analyzed by using Amiview software purchased from Spectral Instruments Imaging (New York, NY).

Isolation of cells from spleen, liver, colon, and Small Intestine
Spleen and liver were mashed through a 70 μm cell strainer, and mononuclear cells (MNC) were isolated with 40% and 70% percoll. Colon and Small Intestine were cut first longitudinally and then laterally into pieces of approximately 0.5 cm length. Tissue pieces were incubated with 20 mL of predigestion solution (1× HBSS) without containing 5 mM EDTA, 5% fetal bovine serum (FBS), 1 mM DTT) for 20 minutes at 37°C under continuous shaking, then passed through 70 μm strainer, and MNC were isolated with 40% and 70% percoll. For In vivo BrdU labeling and Annexin V staining. On day 6 after HCT, T cell proliferation was measured with a single i.p. injection of BrdU (2.5 mg/mouse, 100 mg/g) 3h before tissue harvesting. Analysis of donor T cells for BrdU incorporation was performed according to the manufacturer's instructions (BD Pharmingen). For Annexin V staining, the percentage of Annexin V + cells was assessed by flow cytometry according to the manufacturer's instructions (ThermoFisher,. For mitochondrial staining, cells were stained with MitoSOX, Mitotracker green, Mitotracker Red dye (ThermoFishe, catalog# M36008, M7514, M7512) according to manufacturer's protocol. Intracellular reduced glutathione was assessed by flow cytometry after staining with Thiol Green according to the manufacturer's instructions (Abcam, catalog# ab112132). Flow cytometry analyses were performed with BD LSRFortessa (Franklin Lakes, NJ), data were analyzed with FlowJo software (Tree Star, Ashland, OR). Cell sorting was performed with a BD FACS Aria SORP sorter (Franklin Lakes, NJ) at the City of Hope FACS facility. The sorted cells were used for RNA isolation.

CDR3 sequencing and analysis
Mouse TCR alpha and beta were amplified using Takara's SMARTer mouse TCR kit to prepare sequencing libraries, which were quantified and sequenced on an miSeq sequencer (Illumina) to generate PE300 reads. TCR sequencing data were aligned and processed using miXCR v3.0 with default settings to obtain TCR CDR3 clones. VDJTools v1.1.7 were used to generate a merged CDR3 clone abundance table for all samples include two batches and each batch combined from 3 mice and to calculate the diversity index.

RNA isolation and RNA sequencing
Each sample represents lymphocytes combined from 3 recipients. RNA from equal numbers of sorted H-2K b+ TCRβ + CD4 + and H-2K b+ TCRβ + CD8 + cells were extracted with the RNeasy Mini Kit (Qiagen, Hilden, Germany). Total RNA sequencing was performed and analyzed by the Integrative Genomics Core, City of Hope National Medical Center (Duarte, CA).
For RNA sequencing, RNA concentration was measured by NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA), and RNA integrity was determined with the use of a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Libraries were constructed from 300 ng total RNA for each sample by using KAPA Stranded mRNA-Seq Kit (Kapa Biosystems, Wilmington, MA) with 10 cycles of PCR amplification. Libraries were purified using AxyPrep Mag PCR Clean-up kit (Thermo Fisher Scientific). Each library was quantified by using a Qubit fluorometer (Life Technologies, Carlsbad, CA), and the size distribution was assessed by using a 2100 Bioanalyzer (Agilent, Santa Clara, CA). Cluster generation was done according to the TruSeq SR Cluster Kit V4-cBot-HS (Illumina, San Diego, CA), and sequencing was performed on an Illumina® Hiseq 2500 (Illumina, San Diego, CA) instrument to generate 51 bp single-end reads. Quality control of RNA-Seq reads was performed using FastQC.

Gene Set Enrichment Analysis
Raw sequences were aligned to the mouse reference genome mm10 using STAR aligner v2.7, and the gene level expression of RefSeq annotation were summarized using Htseq-count v0.11. Raw counts were normalized by trimmed mean of M value (TMM) method and differentially expressed genes between groups were identified using the Bioconductor package "edgeR" v3.32.1. Pre-ranked gene set enrichment analysis (GSEA) was applied to examine the Hallmark and KEGG pathways, obtained from "msigbdr" package v7.4.1, that are significantly modulated using the "clusterProfiler" package v3.16.1. The gene list was ranked by -log10 (P value) with signs determined by logFC for each comparison for the GSEA analysis. Enrichment scores (ES) obtained by GSEA were used to compare different Hallmark and KEGG pathways at different time points after transplantation. P values were adjusted by the "BH" algorithm to generate false discovery rate (5).